This invention relates generally to a method and apparatus to coat a substrate with a small volume of fluid. More particularly, the invention relates to a method and apparatus to quickly and uniformly coat a fluid such as reagents or fluids used in DNA array fabrication onto a surface of a solid substrate by providing a pressure differential between substantially parallel surfaces of a fluid-containing distribution member.
Nucleic acid hybridization is a known method for identifying specific sequences of nucleic acids; hybridization involves base-pairing between complementary nucleic acid strands. When single-stranded nucleic acids are used as probes to identify specific target sequences of nucleic acids, probes of known sequences are exposed to and incubated in sample solutions containing sequences to be identified. If a sequence hybridizes to a probe of a known sequence, the sequence is necessarily the specific target sequence. Various aspects of this method have been studied in detail. In essence, all variations allow complementary base sequences to pair and thus form double-stranded stable molecules, and a variety of methods are known in the art to determine whether pairing has occurred, such as those described in U.S. Pat. No. 5,622,822 to Ekeze et al. and U.S. Pat. No. 5,256,535 to Ylikoski et al.
Hybridization of surface-bound probes to solution-based targets is an effective means to analyze a large number of DNA or RNA molecules in parallel. Specific probes of known sequences are attached to the surface of a solid substrate in known locations. The probes are usually immobilized on a solid support having a surface area of typically less than a few square centimeters. The solid support is typically a glass or fused silica slide which has been treated to facilitate attachment of probes. A mobile-phase sample containing labeled targets, e.g., a buffered aqueous solution containing target DNA, is contacted with and allowed to react with the surface. By detecting the labels to determine whether hybridization has occurred at specific locations, it is possible to determine the composition of the sample and the sequences of the unknown targets. Alternatively, target biomolecules may be bound to the surface while labeled probes are contained in the mobile phase. In either case, the hybridization reaction typically takes place over a time period that can be many hours, for a typical sample containing target material in the concentration range in the picomolar domain.
In the preparation of arrays such as those for use in nucleic acid hybridization, reagents may be applied to predetermined locations on the surface of a substrate. Generally, a surface is first cleaned or otherwise prepared by exposure to a fluid containing a reagent. Then, array preparation will involve application of biomolecule-containing fluids at discrete locations. For nucleic-acid probe array preparation, the biomolecule-containing fluid may contain the already-formed probes that can bind with the surface, or a specific nucleotide that will later constitute a portion of a probe that is synthesized in situ on the surface. Then, treatment of a portion of or the entire surface with a different fluid may follow. The steps may be repeated a number of times in situ to prepare the desired array. Once an array of probes is formed on a substrate surface for hybridization with target molecules in a sample fluid, hybridization may be carried out by uniformly exposing the entire substrate surface to the sample fluid.
It is apparent, then, that surface coating by a fluid is an important aspect in array technology, particularly in the field of biomolecular arrays. Important aspects of coating procedures include the amount of fluid used and the rate of throughput. In general, coating procedures should employ only a small quantity of fluid, for a number of reasons. First, the fluid may contain expensive or rare reagents, and waste of such fluids is undesirable. Second, many ordinary reagents that are used in array preparation are toxic, and decreasing their use is desirable in order to lower the risk of human exposure. A high throughput rate also implies that it takes less time to coat each substrate surface, thereby also lowering the risk of human exposure during the coating procedure.
Another important aspect of coating procedures is uniformity of coverage. For biomolecular arrays, it is desirable to uniformly apply a fluid onto a substrate surface to ensure that each feature is attached or formed under similar conditions. In addition, during use of a formed array containing surface-bound probes, uniform distribution of sample fluid to ensure proper hybridization is necessary. Without uniform fluid distribution, resultant hybridization data will be compromised.
One method by which a surface may be coated with a small amount of fluid is through the use of a flow cell assembly. Variations on the use of a flow cell are described in U.S. Pat. No. 4,596,695 to Cottingham and U.S. Pat. No. 5,145,784 to Cox et al. The basic flow cell method typically provides that a cover and substrate are positioned parallel to each other. A gap is thus formed between the cover and the substrate. To control the size of the gap, one or more spacers having a selected height are disposed within the gap. In addition, the cover, the spacers and the substrate are arranged such that a chamber is provided having an inlet channel and an outlet channel. By creating an appropriate pressure gradient between the inlet and outlet channels, fluid fills the chamber by laminar flow, coating the surface of the substrate within the chamber. By controlling the volume in the chamber through the proper selection of the spacer height, the amount of fluid needed to coat the surface can be reduced.
The use of the flow cell method has a number of drawbacks. First, uniform coating requires laminar flow of the fluid. Laminar flow regime generally implies that there is an absolute upper limit to the volumetric rate given the geometry of the chamber. Second, to increase the flow rate of the fluid, the pressure gradient between the inlet and outlet channels must be increased. However, pressure surges that are generated while increasing the pressure gradient tend to cause the flow cell assembly to leak, either at the cover/support interface or at the support/substrate interface. Third, any irregularity in the surface profile of the substrate tends to disrupt laminar flow. As a result, air pockets may be formed and trapped within the flow cell assembly that will interrupt contact between the fluid and the substrate. Thus, while the use of a flow cell tends to lower the amount of reagent fluid waste, the gain in lowered waste is offset by diminishing throughput.
Spin coating may be employed to quickly and uniformly coat a fluid on a substrate. Spin coating is usually performed by dispensing the fluid at or near the center of a substrate. The substrate is spun either during or after the reagent is dispensed such that the fluid spreads radially and outwardly to cover the entire substrate. In this method, the volume needed to cover a surface depends on fluid property, e.g., viscosity and surface tension, and the surface energy of the substrate. When a low energy surface is provided, a relatively large volume fluid is needed to cover the entire surface. Without sufficient volume, applied fluid tends to exhibit clustering behavior and does not cover the entire surface uniformly. Thus, a relatively large amount of fluid is first applied to the surface at a low spin speed to cover the entire surface. Then, the substrate is spun at a higher speed to remove excess fluid. Consequently, while spin coating may be advantageous in terms of high throughput, it is a relatively wasteful technique.
Thus, there is a need to provide a method and apparatus to coat a substrate surface with a small volume of fluid quickly and uniformly without relying on spin coating or an ordinary flow cell.
Accordingly, it is an object of the present invention to overcome the above-mentioned disadvantages of the prior art by providing a method for coating a substrate surface with a small volume of fluid without generating excess waste.
It is another object of the invention to provide such a method wherein the substrate is coated quickly and uniformly.
It is still another object of the invention to provide an apparatus for use in carrying out the aforementioned method.
It is a further object of the invention to provide such an apparatus for use in carrying out the aforementioned method with a plurality of fluids in succession.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.
In one general aspect, then, the present invention relates to a method for applying a fluid onto an application area located on a surface of a solid substrate. The method comprises providing a distribution member having an upper surface and a lower surface, and including a permeable portion having channels of a selected size. The distribution member may be generally planar and is positioned such that the lower surface of the distribution member is in generally opposing spacing relation to the surface of the substrate. The distribution member and the substrate may also be substantially uniformly spaced relation. A predetermined volume of the fluid is dispensed onto the upper surface of the permeable portion of the distribution member such that the fluid penetrates and is retained by the permeable portion of the distribution member. The fluid may penetrate and be retained by the permeable portion of the distribution member under the application of a loading pressure differential between the upper and lower surfaces. In addition, a distributing pressure differential is applied between the upper and lower surfaces generally without using a movable mechanical means designed to spread fluids such as a squeegee such that at least a portion of the fluid passes through the channels of the permeable portion of the distribution member and onto the application area. The application area may contain any array of biomolecules covalently or otherwise attached thereto.
In another aspect, the invention relates to the above method wherein a perimeter spacer is provided and positioned between and in contact with the lower surface of the distribution member and the surface of the solid substrate. Accordingly, the perimeter spacer defines an application volume between the lower surface of the distribution member and the surface. The fluid passes through the channels of the permeable portion of the distribution member and into the application volume.
In still another aspect, the invention relates to the above method wherein the distribution pressure differential is generated by raising the pressure at the second surface of the distribution member. The distribution pressure differential may also be generated by lowering the pressure at the lower surface of the distribution member.
In a further aspect, the invention relates to the above method further comprising the step of dispensing a second fluid onto the upper surface of at least the permeable portion of the distribution member. A pressure differential between the upper and lower surfaces is applied such that the second fluid passes through the channels in the permeable portion of the distribution member and onto the application area, displacing the fluid away from the solid substrate surface. In addition, the method may further comprise the step of flushing a gas through the permeable portion of the distribution member such that the second fluid is displaced away from the solid substrate surface. The gas may comprise nitrogen or argon.
In a still further aspect, the invention relates to the above method wherein the fluid contains water, or an organic solvent such as acetonitrile, an alcohol, or a ketone. Likewise, the fluid may comprise a biomolecule. Examples of biomolecules include oligonucleotides, polynucleotides, oligopeptides and polypeptides. In order to prevent fluid waste, it is preferred that the predetermined volume of the fluid does not substantially exceed the application volume. More preferably, the predetermined volume of the fluid should not exceed about 150% of the application volume. Still more preferably, the predetermined volume should not exceed about 110% of the application volume.
In another general aspect, the invention relates to an apparatus for applying a fluid onto a surface of a solid substrate. The apparatus comprises a distribution member having an upper surface, a lower surface and a permeable portion formed by a plurality of channels extending from the upper surface to the lower surface. Such a distribution member may comprise a perforated flat piece or a mesh. Affixed in sealed contact with the upper surface about the permeable portion of the distribution member is an enclosing wall that, together with the distribution member, defines an enclosure having an enclosure volume. The apparatus also provides means for positioning the distribution member in relation to the solid substrate surface such that the lower surface of the distribution member is in generally uniformly spaced opposing relationship to the solid substrate. Once fluid is introduced onto the fluid distribution area within the enclosure, a means for producing a positive pressure differential between the upper surface and the lower surface of the distribution member can be activated. As a result, the liquid passes through the member and onto the solid substrate surface.
In another aspect, the invention relates to the above apparatus wherein a spacer having generally parallel upper and lower surfaces is provided. In such a case, the upper surface of the spacer is affixed to the lower surface of the distribution member about the permeable portion. The lower surface is placed in contact with the substrate such that an application space having an application volume is substantially enclosed by the spacer, the lower surface of the distribution member, and the substrate surface. An opening may be disposed in the spacer such that the application space fluidly communicates with open air. In order to prevent fluid waste, it is preferred that the enclosure volume does not substantially exceed the application volume. More preferably, the enclosure volume should not exceed about 150% of the application volume. Still more preferably, the enclosure volume should not exceed about 110% of the application volume.
In still another aspect, the invention relates to the above apparatus wherein the means for introducing the fluid comprises a fluid source for supplying the fluid. A fluid transfer channel is connected to the enclosure and, a fluid valve is disposed between the fluid source and the fluid transfer channel. Fluid communication is provided from the fluid source to the fluid transfer channel when the fluid valve is open. In addition, the apparatus may include means for introducing a second fluid. Such means may comprise a second fluid source for supplying the second fluid and a second fluid valve disposed between the second fluid source and the fluid transfer channel, where the second fluid source is in fluid communication with the fluid transfer channel when the second fluid valve is open. In any case, the fluid may contain a liquid such as water, acetonitrile, an alcohol, or a ketone for use in facilitating chemical reactions. Where hybridization reactions are desired, the fluid will contain a biomolecule such as an oligonucleotide, polynucleotide, oligopeptide, or polypeptide.
In a further aspect, the invention relates to the above apparatus wherein the means for producing a pressure differential may comprise means for raising pressure within the chamber. Such pressure raising means may comprise means for introducing a gas into the enclosure from a pressured source. The gas may comprise, for example, nitrogen or argon.
In still another general aspect, the invention relates to a reagent application station for applying a plurality of reagents onto a surface of a solid substrate. The apparatus comprises a distribution member having an upper surface, a lower surface and a permeable portion formed by a plurality of channels extending from the upper surface to the lower surface. An enclosure is formed by the upper surface of the distribution member and an enclosing wall affixed about the permeable portion in sealed contact with the upper surface of the distribution member. The apparatus also provides means for positioning the distribution member in relation to the solid substrate surface such that the lower surface of the distribution member is in generally uniformly spaced opposition relation to the solid substrate. To control pressure within enclosure, a variable pressure pump is provided having an inlet for each reagent and an outlet in fluid communication with the enclosure. To supply the reagents, a source for each reagent is provided, and a valve is disposed between each inlet and source to provide individual control over fluid dispensing. It is preferred that no two valves are open at the same time.